JPS6024718B2 - Method for producing highly pure and low colored ribonucleic acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing ribonucleic acid (hereinafter referred to as RNA).

RNA is a substance useful as a raw material for seasonings, cosmetics, pharmaceuticals, etc., and RNA used for such purposes is required to be a product of high purity and low degree of coloration. The present invention involves the production of RNA in which a protease is applied to an RNA-containing solution to decompose proteins present as impurities, and then decolorized. The present invention relates to a method for industrially easily and inexpensively producing RNA with high purity and low degree of coloring, which is characterized by removing colored components with a decolorizing agent. There are many extraction methods for extracting RNA from microbial cells, such as hot saline extraction, weak alkaline heating extraction, or extraction after mechanically crushing or enzymatically lysing the microbial cells. RNA obtained by extraction method
The extract usually contains large amounts of impurities such as proteins, sugars, and salts. As a method for recovering RNA from this RNA extract on an industrial scale, RN is generally recovered under acidic conditions.
A method is used to insolubilize A and separate and recover it. However, among the proteins present as impurities, acid-insoluble proteins or proteins bound to nucleic acids behave in the same way as RNA, and it is difficult to separate and remove them from RNA using the methods described above. Therefore, RNA products obtained by such RNA separation and recovery methods have relatively low purity. An object of the present invention is to provide an industrial method for producing highly pure RNA by separating and removing such proteins. By the way, known methods for obtaining highly pure RNA by separating and removing proteins include a method in which proteins are partially denatured by phenol treatment and extracted into a phenol layer, and a method in which proteins are denatured and removed with chloroform. .

However, these methods use large amounts of organic solvents and
Furthermore, since the operation is complicated, it is difficult to say that it is suitable for industrial production. In the present invention, first, as a method for efficiently removing proteins, a method using a proteolytic enzyme was adopted because it is optimal in terms of cost and ease of operation.

In other words, by allowing a protease to act on an RNA-containing solution, acid-resistant proteins are degraded to make them acid-soluble, and proteins bound to RNA are degraded. It was discovered that the binding could be broken by decomposition, and that the protein could be easily separated and removed during the subsequent separation and recovery of RNA by acid precipitation. Table 1 shows the protein removal effects of proteolytic enzymes. Table 1. However, although this method achieves the purpose of protein removal, the resulting RNA has a high degree of coloration, whereas RNA with a low degree of coloration
It was found that in order to obtain A, a decolorization operation was necessary.

Furthermore, as shown in Table 2, when proteolytic enzyme treatment is performed, browning of the liquid is observed as a side reaction during the reaction, which is thought to be caused by an amino-carbonyl reaction. etc., it is extremely difficult to remove. In other words, it has been found that even if an RNA solution highly purified using a proteolytic enzyme is directly decolored with a decolorizing agent such as activated carbon, the desired RNA with a low degree of coloration cannot be obtained. Therefore, the high purity table of RNA-2 note, the degree of coloring is 5%.
The absorbance at a wavelength of 450 m of the supernatant obtained by centrifuging the RNA solution at 10,000 m blood for 10 minutes was measured, and the value was expressed as coloring degree = absorbance x 1000.

As a result of intensive research into a method for efficiently obtaining highly pure and low-color RNA that can be easily decolorized by subsequent decolorization using a decolorizing agent, the method using proteolytic enzymes, which is the most efficient method for chemical reaction, can be adopted. discovered that this object could be achieved by adding one or more of sulfite, bisulfite, or a salt thereof as a coloring preventive agent in advance from the stage of action of proteolytic enzymes, which led to the present invention. It is something that

Table 3 shows an example of using sodium bisulfite as a coloring inhibitor. Table 3 By the way, when using activated carbon, which has the greatest decolorizing effect among various decolorizing agents, in decolorizing an RNA-containing solution, it is difficult to completely remove the added activated carbon due to the decomposition phenomenon of activated carbon, which is a characteristic of colloidal solutions. Since it cannot be removed and some of it is carried into the RNA, it is difficult to obtain RNA with a satisfactory low degree of coloration.

However, according to the method in which the decolorizing agent of the present invention is used from the protein decomposition stage, the RNA-containing solution is in a state where it is extremely easy to decolorize. It can be easily decolored with exchangeable resin, etc., and even when powdered activated carbon is used, only a relatively small amount is required, so it is possible to minimize leakage of activated carbon due to the decomposition phenomenon. , it became possible to obtain RNA with a low degree of coloration extremely easily. As described above, the feature of the present invention is that, as a result of intensive investigation into an industrially viable production method for highly pure and low-color RNA, protein existing as an impurity is removed by applying a protease to an RNA-containing solution. RN decomposes and then decolorizes
In the production of A, a coloring inhibitor is added in advance at the stage where a proteolytic enzyme is applied, and the colored components are removed using a decolorizing agent after the proteolytic reaction.

In the present invention, each operation is not simply an accumulation of effects, but is organically linked to each other, and as shown in Table 4, even if any of the operations is missing, RNA of the desired quality can be obtained.
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In other words, the present invention establishes an extremely efficient and economical industrial production method for RNA with a quality equal to or higher than that of high-quality RNA that could previously only be obtained by using large amounts of organic solvents and repeating complicated operations. This is what makes it so special. Table 4 Note that as the RNA-containing solution, it is also possible to use an extract obtained from microbial cells or plant cells, or a solution obtained by redissolving RNA separated from them in water.

Further, as a protease, it can be used regardless of its origin and degree of purification.

Activated carbon and ion exchange resin used as decolorizing agents have different decolorizing effects depending on their type, but any type can be used immediately.

Example 1 Yeast cells (Candidautilis) 10k9 obtained by culturing sulfite pulp waste liquid as a sugar source were suspended in 100 g of a 5% saline solution, heated at 95 qo for 4 hours, and then extracted with yeast cells by centrifugation. The body was removed to obtain 100ml of RNA-containing liquid.

Sodium hydrogen sulfite was added to this solution for 10 minutes, and 2.5 g of Bronase (manufactured by Kaken Kagaku KK) was added as a proteolytic enzyme.
After reacting at 50° C. for 1 hour, the solution was cooled to 500° C., and the pH was adjusted to 1.5 using hydrochloric acid to insolubilize the RNA. The insolubilized RNA is separated and collected by centrifugation, then suspended in water again, neutralized with sodium hydroxide, and R
20% of NA-containing liquid was obtained. Next, add this liquid to granular activated carbon (
Granular white heron (manufactured by Takeda Pharmaceutical Co., Ltd. KK) was passed through a column packed with 100 ml of decolorized liquid at SV = 2.
High purity and low coloring degree RNA1[9 with an NA purity of 96.5% and a coloring degree of 38 was obtained. Example 2 Yeast cells (Sa) obtained by culturing molasses as a sugar source
ccharomyces cerevisiae)lk9
was suspended in a 4% saline solution for 10 minutes, heated at 98°C for 2 hours, and then the bacterial cells were removed by centrifugation.
Ten volumes of the containing liquid were obtained.

Claims (1)

[Claims] 1. In the production of ribonucleic acid, in which a protease is applied to a solution containing ribonucleic acid to decompose proteins present as impurities and then decolorized, sulfite, bisulfite, or High purity and low coloring degree ribonucleic acid is produced by adding one or more selected from the group consisting of sodium salts or potassium salts thereof, and removing colored components with a decolorizing agent after proteolytic reaction. Production method.